Inhibitors of prototypic galectin dimerization and uses thereof

ABSTRACT

Agents that inhibit the dimerization of a prototypic galectin such as galectin-7 are described. These agents, for example antibodies and peptides, bind to a domain corresponding to residues 13-25, 86-108 and/or 129-135 of human galectin-7. The use of such agents to inhibit a biological, physiological and/or pathological process that involves prototypic galectin dimerization, for example for the inhibition of galectin-7-mediated apoptosis and the treatment of galectin-7-expressing cancers, is also described.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Entry Application of PCTapplication No. PCT/CA2016/050587, filed on May 25, 2016, which claimsthe benefits of U.S. provisional application Ser. No. 62/167,512, filedon May 28, 2015, which are incorporated herein by reference in theirentirety.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R. §1.821, entitled 9355-8_ST25.txt, 12,687 bytes in size, generated on Nov.22, 2017 and filed via EFS-Web, is provided in lieu of a paper copy.This Sequence Listing is hereby incorporated by reference into thespecification for its disclosures.

TECHNICAL FIELD

The present invention generally relates to prototypic galectins, andmore particularly to the inhibition of galectin-7 dimerization andrelated applications.

BACKGROUND ART

Cancer is a complex pathology manifested by uncontroIled growth of cellsthat have undergone various transformations from physiologically normalcells. Several hallmarks provide a methodical and rational approach instudying this disease, namely the sustaining of proliferative signaling,evasion of growth suppressors, resistance to cell death, replicativeimmortality, angiogenesis, activation of invasion, and metastasis [1].In recent years, however, strong evidence has highlighted the importantrole of immune cells present in the tumor micro-environment [2, 3]. Forinstance, one way that tumor cells can modulate and escape immunedestruction is by secretion of various factors such as pro-inflammatoryeicosanoids, cytokines, chemokines and other soluble signaling moleculesleading to the formation of an immunosuppressive tumor micro-environment[4].

Galectins are multifunctional proteins belonging to the animal lectinfamily. All galectins share similar binding affinities to β-galactosidesand display high amino acid sequence homology among theircarbohydrate-binding domains (CRDs) [5]. In mammals, 19 differentmembers have been identified, and 13 of them have been identified inhumans. Galectins are divided in three sub-groups according to theirstructure: prototypic galectins containing one CRD (Gal-1, -2, -5, -7,-10, -13, -14, -15, -16, -17, -19, and -20), tandem-repeat galectinscontaining two-CRDs covalently linked (Gal-4, -6, -8, -9 and -12) and achimera-type galectin containing multiple CRDs linked by theiramino-terminal domain (Gal-3) [6, 52, 53]. While these proteins performhomeostatic functions inside normal cells, under pathological or stressconditions, cytosolic galectins are released either passively from deadcells or actively via non-classical secretion pathways [7]. Once in theextracellular milieu, they bind all glycosylated growth receptors on thesurface of normal and cancer cells to set their signaling threshold [8,9]. Such properties enable galectins to kill infiltrating immune cellswhile promoting growth of tumour cells [9]. Galectins are thus idealtargets for effective therapeutics, and new approaches are thereforebeing developed to modulate their activities [10]. These avenues havefocused mainly on carbohydrate-based inhibitors disrupting extracellulargalectins, which form multivalent complexes with cell surfaceglycoconjugates to deliver CRD-dependent intracellular signals thatmodulate cell activation and survival/apoptosis. Despite decades ofresearch, however, the progression in this field has been very slow. Inmost cases, these inhibitors are high molecular weight, naturallyoccurring polysaccharides that are used to specifically block thebinding of extracellular galectins to carbohydrate structures [11-14].Unfortunately, such inhibitors often display low affinity, lack ofselectivity for a given galectin due to highly conserved homology amonggalectin CRDs, and are not effective at targeting CRD-independentfunctions of galectins. Indeed, several studies have shown that severalcritical biological processes of galectins are mediated viaCRD-independent interactions [15-18].

There is thus a need for novel modulators of galectins, for exampleinhibitors that targets CRD-independent functions of galectins.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

The present invention provides the following items 1 to 52:

1. A peptide or peptidomimetic of 50 residues of less that inhibitshuman galectin-7 dimerization, said peptide comprising:

(i) a domain comprising at least 5 residues of the sequence of formulaII:Xaa¹⁴-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Xaa¹⁹-Xaa²⁰  (II)wherein“-” represents a bond;Xaa¹⁴ is L-Leu or D-Leu;Xaa¹⁵ is L-Asp or D-Asp;Xaa¹⁶ is L-Ser or D-Ser;Xaa¹⁷ is L-Val or D-Val;Xaa¹⁸ L-Arg or D-Arg;Xaa¹⁵ is L-Ile or D-Ile;Xaa²⁰ is L-Phe or D-Phe;or a domain comprising at least 5 residues of the sequence of formula IIin which one of Xaa¹⁴, Xaa¹⁶, Xaa¹⁷, Xaa¹⁸, Xaa¹⁶ or Xaa²⁰ is mutated;(ii) a domain comprising at least 5 residues of the sequence of formulaI:Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³  (I)“-” represents a bond;Xaa¹ is L-Ile or D-Ile;Xaa² is L-Arg or D-Arg;Xaa³ is L-Pro or D-Pro;Xaa⁴ is Gly;Xaa⁵ is L-Thr or D-Thr;Xaa⁶ is L-Val or D-Val;Xaa⁷ is L-Leu or D-Leu;Xaa⁸ is L-Arg or D-Arg;Xaa⁹ is L-Ile or D-Ile;Xaa¹⁰ is L-Arg or D-Arg;Xaa¹¹ is Gly;Xaa¹² is L-Leu or D-Leu;Xaa¹³ is L-Val or D-Val;or a domain comprising at least 5 residues of the sequence of formula Iin which 1 or 2 residue(s) is/are mutated; or(iii) a domain comprising at least 5 residues of the sequence of formulaIII:Xaa²¹-Xaa²²-Xaa²³-Xaa²⁴-Xaa²⁵-Xaa²⁶-Xaa²⁷-Xaa²⁸-Xaa²⁹-Xaa³⁰-Xaa³¹-Xaa³²-Xaa³³-Xaa³⁴-Xaa³⁵-Xaa³⁶-Xaa³⁷-Xaa³⁸-Xaa³⁹-Xaa⁴⁰-Xaa⁴¹-Xaa⁴²-Xaa⁴³  (III)wherein “-” is a bond;Xaa²¹ is L-Phe or D-Phe;Xaa²² is L-Glu or D-Glu;Xaa²³ is L-Val or D-Val;Xaa²⁴ is L-Leu or D-Leu;Xaa²⁵ is L-Ile or D-Ile;Xaa²⁶ is L-Ile or D-Ile;Xaa²⁷ is L-Ala or D-Ala;Xaa²⁸ is L-Ser or D-Ser;Xaa²⁹ is L-Asp or D-Asp;Xaa³⁰ is L-Asp or D-Asp;Xaa³¹ is Gly;Xaa³² is L-Phe or D-Phe;Xaa³³ is L-Lys or D-Lys;Xaa³⁴ is L-Ala or D-Ala;Xaa³⁵ is L-Val or D-Val;Xaa³⁶ is L-Val or D-Val;Xaa³⁷ is Gly;Xaa³⁸ is L-Asp or D-Asp;Xaa³⁹ is L-Ala or D-Ala;Xaa⁴⁰ is L-Gln or D-Gln;Xaa⁴¹ is L-Tyr or D-Tyr;Xaa⁴² is L-His or D-His, andXaa⁴³ is L-His or D-His;or a domain comprising at least 5 residues of the sequence of formulaIII in which 1 or 2 residue(s) is/are mutated.or a salt thereof.2. The peptide, peptidomimetic or salt thereof of item 1, whichcomprises a domain comprising at least 5 residues of the sequence offormula II, or a domain of formula II in which one of Xaa¹⁴, Xaa¹⁶,Xaa¹⁷, Xaa¹⁸, Xaa¹⁹ or Xaa²⁰ is mutated.3. The peptide, peptidomimetic or salt thereof of item 2, whichcomprises a domain comprising at least 5 residues of the sequence offormula II, or a domain of formula II in which one of Xaa¹⁶ or Xaa¹⁸ ismutated.4. The peptide, peptidomimetic or salt thereof of item 3, whichcomprises a domain comprising at least 5 residues of the sequence offormula II.5. The peptide, peptidomimetic or salt thereof of any one of items 2 to4, which has 20 residues or less.6. The peptide, peptidomimetic or salt thereof of item 5, which has 15residues or less.7. The peptide, peptidomimetic or salt thereof of item 6, which has 10residues or less.8. The peptide, peptidomimetic or salt thereof of item 7, which has 7residues.9. The peptide, peptidomimetic or salt thereof of any one of items 1 to8, which comprises the following domain: Leu-Asp-Ser-Val-Arg-Ile-Phe(SEQ ID NO:1).10. The peptide, peptidomimetic or salt thereof of item 1, whichcomprises a domain comprising at least 5 residues of the sequence offormula I, or a domain of formula I in which 1 or 2 residue(s) is/aremutated.11. The peptide, peptidomimetic or salt thereof of item 10, whichcomprises a domain comprising at least 5 residues of the sequence offormula I, or a domain of formula I in which 1 residue is mutated.12. The peptide, peptidomimetic or salt thereof of item 10, whichcomprises a domain comprising at least 5 residues of the sequence offormula I.13. The peptide, peptidomimetic or salt thereof of any one of items 10to 12, which has 20 residues or less.14. The peptide, peptidomimetic or salt thereof of item 13, which has 15residues or less.15. The peptide, peptidomimetic or salt thereof of item 14, which has 13residues.16. The peptide, peptidomimetic or salt thereof of any one of items 10to 15, which comprises the following domain:Ile-Arg-Pro-Gly-Thr-Val-Leu-Arg-Ile-Arg-Gly-Leu-Val (SEQ ID NO:3).17. The peptide, peptidomimetic or salt thereof of item 1, whichcomprises a domain comprising at least 5 residues of the sequence offormula IIIA or IIIB:Xaa²¹-Xaa²²-Xaa²³-Xaa²⁴-Xaa²⁵-Xaa²⁶-Xaa²⁷-Xaa²⁸-Xaa²⁹-Xaa³⁰-Xaa³¹-Xaa³²-Xaa³³-Xaa³⁴-Xaa³⁵-Xaa³⁶-Xaa³⁷  (IIIA):Xaa³⁰-Xaa³¹-Xaa³²-Xaa³³-Xaa³⁴-Xaa³⁵-Xaa³⁶-Xaa³⁷-Xaa³⁸-Xaa³⁹-Xaa⁴⁰-Xaa⁴¹-Xaa⁴²-Xaa⁴³  (IIIB):or a domain comprising at least 5 residues of the sequence of formulaIIIA or IIIB in which 1 or 2 residue(s) is/are mutated.wherein “-” and Xaa²¹ to Xaa⁴³ are as defined above.18. The peptide, peptidomimetic or salt thereof of item 17, which has 20residues or less.19. The peptide, peptidomimetic or salt thereof of item 18, which has 15to 20 residues.20. The peptide, peptidomimetic or salt thereof of any one of items 10to 15, which comprises the following domain:Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-Asp-Ala-Gln-Tyr-His-His (SEQ ID NO:5) orPhe-Glu-Val-Leu-Ile-Ile-Ala-Ser-Asp-Asp-Gly-Phe-Lys-Ala-Val-Val-Gly (SEQID NO:7).21. The peptide, peptidomimetic or salt thereof of any one of items 1 to17, which is of the following formula III:Z¹-D-Z²whereinZ¹ is H or is an amino-terminal modifying group;D is the domain of formula I, II or III defined in any one of items 1 to20; andZ² is OH or is a carboxy-terminal modifying group;22. The peptide, peptidomimetic or salt thereof of item 21, wherein saidamino-terminal modifying group is (i) an acyl group (R—CO), wherein R isa hydrophobic moiety, or (ii) an aroyl group (Ar—CO), wherein Ar is anaryl group.23. The peptide, peptidomimetic or salt thereof of item 21, wherein Z¹is H.24. The peptide, peptidomimetic or salt thereof of any one of items 21to 23, wherein said carboxy-terminal modifying group is a hydroxamategroup, a nitrile group, an amide group, an alcohol or CH₂OH.25. The peptide, peptidomimetic or salt thereof of item 24, wherein Z²is NH₂.26. The peptide, peptidomimetic or salt thereof of item 25, which is ofone of the following sequences: Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂ (SEQ IDNO:2), Ile-Arg-Pro-Gly-Thr-Val-Leu-Arg-Ile-Arg-Gly-Leu-Val-NH₂ (SEQ IDNO:4), Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-Asp-Ala-Gln-Tyr-His-His-NH₂ (SEQID NO:6) orPhe-Glu-Val-Leu-Ile-Ile-Ala-Ser-Asp-Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-NH₂(SEQID NO:8).27. The peptide, peptidomimetic or salt thereof of item 25, wherein saidpeptide, peptidomimetic or salt thereof is conjugated to a polyethyleneglycol (PEG) chain.28. A composition comprising the peptide, peptidomimetic or salt thereofof any one of items 1 to 27, and a carrier or excipient.29. A method for inhibiting the dimerization of native prototypicgalectin polypeptides, said method comprising contacting said nativeprototypic galectin polypeptides with an agent that binds to a domaincorresponding to residues 13-25, 86-108 and/or 129-135 of humangalectin-7.30. The method of item 29, wherein said agent is an antibody thatspecifically binds to an epitope located within a domain correspondingto residues 13-25, 86-108 and/or 129-135 of human galectin-7.31. The method of item 30, wherein said agent is an antibody thatspecifically binds to an epitope located within residues 129-135 ofhuman galectin-7.32. The method of item 29, wherein said agent is the peptide,peptidomimetic or salt thereof of any one of items 1 to 26.33. A method for inhibiting galectin-7-mediated apoptosis in a cell,said method comprising contacting said cell with the agent defined inany one of items 29 to 32.34. The method of item 33, wherein said cell is an immune cell.35. The method of item 34, wherein said immune cell is a T lymphocyte.36. A method for treating a prototypic galectin-expressing cancer in asubject, said method comprising administering to said subject aneffective amount of the agent defined in any one of items 29 to 32.37. The method of item 36, wherein said prototypic galectin-expressingcancer is a galectin-7-expressing cancer.38. The method of item 37, wherein said galectin-7-expressing cancer isa breast cancer, an ovarian cancer, or a lymphoma.39. Use of the agent defined in any one of items 29 to 32 for inhibitingthe dimerization of native galectin-7 polypeptides.40. Use of the agent defined in any one of items 29 to 32 for themanufacture of a medicament for inhibiting the dimerization of nativegalectin-7 polypeptides.41. Use of the agent defined in any one of items 29 to 32 for inhibitinggalectin-7-mediated apoptosis in a cell.42. Use of the agent defined in any one of items 29 to 32 for themanufacture of a medicament for inhibiting galectin-7-mediated apoptosisin a cell.43. The use of item 41 or 42, wherein said cell is an immune cell.44. The use of item 43, wherein said immune cell is a T lymphocyte.45. Use of the agent defined in any one of items 29 to 32 for treating aprototypic galectin-expressing cancer in a subject.46. Use of the agent defined in any one of items 29 to 32 for themanufacture of a medicament for treating a prototypicgalectin-expressing cancer in a subject.47. The use of item 45 or 46, wherein said prototypicgalectin-expressing cancer is a galectin-7-expressing cancer.48. The use of item 47, wherein said galectin-7-expressing cancer is abreast cancer, an ovarian cancer, or a lymphoma.49. A method for determining whether a test agent that may be used toinhibit a biological, physiological and/or pathological process thatinvolves prototypic galectin dimerization, said method comprising:

contacting a prototypic galectin polypeptide with said test agent; and

determining whether said test agent binds to prototypic galectinpolypeptide through a domain corresponding to residues 13-25, 86-108and/or 129-135 of galectin-7,

wherein the binding of said test agent to said prototypic galectinpolypeptide is indicative that said test agent that may be used toinhibit a biological, physiological and/or pathological process thatinvolves prototypic galectin dimerization.

50. The method of item 49, wherein said prototypic galectin isgalectin-7.

51. The method of item 49 or 50, wherein said method comprisesdetermining whether said test agent binds to said prototypic galectinpolypeptide through a domain corresponding to residues 129-135 ofgalectin-7.

52. The method of any one of items 49 to 51, wherein said method is fordetermining whether the test agent may be used to inhibitgalectin-7-mediated apoptosis in a cell and/or treat agalectin-7-expressing cancer in a subject.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

In the appended drawings:

FIGS. 1A to C show the dimeric structure of hGal-7. FIG. 1A Dimerformation of recombinant hGal-7 and hGal-1 at increasing concentrationswere compared by polyacrylamide gel electrophoresis in low-sodiumdodecyl sulfate (SDS) conditions. FIG. 1B: Structural representation ofthe hGal-7 (PDB 1BKZ) and hGal-1 (PDB 3W58) dimers with residues 129-135highlighted (balls and sticks) on the hGal-7 dimer interface. Dimerformation in hGal-7 proceeds through a “back-to-back” topology of themonomers while hGal-1 adopts a “side-by-side” structural arrangement,affording additional specificity for galectin inhibition. FIG. 1C:Molecular interactions implicated in the wild-type hGal-7 dimerinterface between residues 129-135 of the first hGal-7 monomer andfacing residues on the second hGal-7 monomer (PDB 1BKZ). Hydrogenbonding and electrostatic interactions are identified as dashed lines.The side chain of Phe135 is also involved in a number of van der Waalsinteractions. The structures were prepared using PyMOL.

FIGS. 2A to 2D show the dose-dependent disruption of the hGal-7 dimer inthe presence of hGal-7₍₁₃₋₂₅₎, hGal 7₍₈₅₋₁₀₂₎, hGal-7₍₉₅₋₁₀₈₎ orhGal-7₍₁₂₉₋₁₃₅₎ peptides. FIGS. 2A and 2B: The recombinant hGal-7 (0.5μM) was incubated with increasing concentrations of hGal-7₍₁₂₉₋₁₃₅₎(FIG. 2A), hGal-7₍₁₃₋₂₅₎, hGal-7₍₈₅₋₁₀₂₎ or hGal-7₍₉₅₋₁₀₈₎ (FIG. 2B) in20 mM potassium phosphate buffer (pH 7.1). FIG. 2C: Incubation of therecombinant hGal-1 and hGal-7₍₁₂₉₋₁₃₅₎ was performed in the samepotassium phosphate buffer. The effect of hGal-7₍₁₂₉₋₁₃₅₎ on themonomeric and dimeric forms of hGal-7/hGal-1 was assessed by Westernblotting in in low-SDS conditions with respective antibodies. The hGal-1film was overexposed. PACAP₂₈₋₃₈ was the control peptide used in orderto ensure the specificity of hGal-7₍₁₂₉₋₁₃₅₎. FIG. 2D: RecombinanthGal-7 (0.5 μM) was also incubated with increasing concentrations ofhGal-7₍₁₂₉₋₁₃₅₎ in 0.1 mM lactose solution.

FIG. 2E: The recombinant hGal-7 (0.5 μM) and Bcl-2 were incubated withincreasing concentrations of hGal-7₍₁₂₉₋₁₃₅₎ in 20 mM potassiumphosphate buffer (pH 7.1). The effect of hGal-7₍₁₂₉₋₁₃₅₎ on thehomodimerization and heterodimerization (with Bcl-2) of hGal-7 wasassessed by Western blotting in low-SDS conditions with an anti-Gal-7antibody. Results depicted in FIGS. 2A to 2D are representative of threeindependent experiments.

FIG. 3 shows that biotin-labeled hGal-7₍₁₂₉₋₁₃₅₎ is capable of bindingto recombinant hGal-7. Binding curve showing a dose-dependentinteraction between biotin-labeled hGal-7₍₁₂₉₋₁₃₅₎ and hGal-7 or hGal-1.Recombinant hGal-7 or hGal-1 (10 μg/ml) were coated on 96-well platesovernight and then incubated 60 min with unlabeled hGal-7₍₁₂₉₋₁₃₅₎ (1mM) to eliminate non-specific binding. Incubation with increasingconcentrations of biotin-labeled hGal-7₍₁₂₉₋₁₃₅₎ was performed for 120min. Results are representative of three independent experiments. Errorbars represent standard deviation.

FIGS. 4A and 4B show the increased binding of hGal-7 on Jurkat T cellsdue to increasing concentrations of hGal-7₍₁₂₉₋₁₃₅₎. FIG. 4A: Histogramshowing the mean fluorescence intensities (MFI) of cells due tofluorescein isothiocyanate (FITC)-labeled hGal-7 binding. FIG. 4B: Flowcytometry histogram displaying the fluorescence (FL1) of the cellpopulation due to FITC-labeled hGal-7 binding. Recombinant hGal-7conjugated to FITC (0.1 μM) was pre-incubated with hGal-7₍₁₂₉₋₁₃₅₎.Jurkat T cells were then harvested in PBS (sodium-azide 0.01%) andincubated for 30 min with their respective dilutions before flowcytometry analysis. Results are representative of three independentexperiments. Error bars represent standard deviation.

FIGS. 5A to 5C show that the apoptotic levels of Jurkat T cells inducedby hGal-7 were decreased due to the presence of hGal-7₍₁₂₉₋₁₃₅₎. FIG.5A: Recombinant hGal-7 was pre-incubated with the respective peptideconcentrations prior to its addition to Jurkat T cells for 4 h at 37° C.in RPMI serum-free media. Apoptosis was monitored by measuring Parp-1cleavage through Western blotting. FIG. 5B: The peptide PACAP₂₈₋₃₈ wasused as a control to ensure the specificity of hGal-7₍₁₂₉₋₁₃₅₎. Flowcytometry histogram showing Annexin V (AV) (FL1) and propidium iodide(PI) (FL3) labeling of Jurkat T cells in the presence of hGal-7 with orwithout hGal-7₍₁₂₉₋₁₃₅₎ treatments. Cells in the lower right quadrantare representative of AV-positive, early apoptotic cells. Cells in theupper right quadrant indicate AV-positive/PI-positive, late apoptoticcells. FIG. 5C: Histogram showing the average percentage of AV positiveJurkat T cells was obtained by adding the percentage of cells found inthe lower and the upper right quadrants. Results are representative ofthree independent experiments. Error bars represent standard deviation.

FIGS. 6A to 6C show the visualization of the dimer-monomer equilibriumof hGal-7 in electrophoretic polyacrylamide gel in low SDS conditions.Recombinant hGal-7 (4 μg) was incubated with increasing SDSconcentrations (%). hGal-7 was then migrated in a SDS-free gel and 0.1%SDS running buffer for 1h, at 150V (FIG. 6A) and in a SDS-free gel andrunning buffer for 4 h, at 100V (FIG. 6B). FIG. 6C: to ensure denaturingconditions, hGal-7 was also migrated in a 0.1% SDS gel and 0.1% SDSrunning buffer for 1 h at 150 V, while the protein was treated withβ-mercaptoethanol and heated for 10 min at 95° C., prior to loading thegel.

FIGS. 7A and 7B show the disruption of hGal-7 dimer by increasingconcentrations of the hGal-7₍₁₂₉₋₁₃₅)-biotin peptide. FIG. 7A:Recombinant hGal-7 (0.5 μM) was incubated with increasing concentrationsof hGal-7₍₁₂₉₋₁₃₅₎ in 20 mM potassium phosphate buffer (pH 7.1). Westernblotting in low-SDS conditions assessed dimer disruption of hGal-7. FIG.7B: Recombinant hGal-1 (0.5 μM) was incubated with increasingconcentrations of hGal-7₍₁₂₉₋₁₃₅₎ in 20 mM potassium phosphate buffer(pH 7.1). Western blotting in low-SDS conditions assessed dimerdisruption of hGal-1. Results are representative of two independentexperiments.

FIGS. 8A to 8D show that peptides hGal-7₍₁₂₉₋₁₃₅₎ and PACAP₂₈₋₃₈ do notinduce toxicity in Jurkat T cells. FIG. 8A: Flow cytometry histogramshowing propidium iodide (PI) (FL3) labeling of Jurkat T cells withoutor with 200 μM of hGal-7₍₁₂₉₋₁₃₅₎. FIG. 8B: Histogram showing theaverage percentage of Annexin-V (AV) positive Jurkat T cells in thepresence of increasing concentrations of hGal-7₍₁₂₉₋₁₃₅₎. FIG. 8C: Flowcytometry histogram showing PI (FL3) labeling of Jurkat T cells withoutor with 200 μM of PACAP₂₈₋₃₈. FIG. 8D: Histogram showing the averagepercentage of AV-positive Jurkat T cells in the presence of increasingconcentrations of PACAP₂₈₋₃₈. The percentage of cell death was obtainedas described above. Error bars represent standard deviation.Significance was calculated with respect to AF samples. Results arerepresentative of three independent experiments.

FIGS. 9A to 9E show that Ala-scan mutants of hGal-7₍₁₂₉₋₁₃₅₎ differ intheir ability to disrupt hGal-7 dimer formation. Disruption of hGal-7dimer was measured in response to increasing concentrations ofhGal-7₍₁₂₉₋₁₃₅₎ (FIG. 9A) and its various Ala-scan mutants:[Ala¹³⁰]hGal-7₍₁₂₉₋₁₃₅₎ (FIG. 9B); [Ala¹³¹]hGal-7₍₁₂₉₋₁₃₅₎ (FIG. 9C);[Ala¹³³hGal-7₍₁₂₉₋₁₃₅₎ (FIG. 9D); and [Ala¹³⁵]hGal-7₍₁₂₉₋₁₃₅₎ (FIG. 9E).The recombinant hGal-7 (0.5 μM) was incubated with increasingconcentrations of hGal-7₍₁₂₉₋₁₃₅₎ peptides. The effect on the monomericand dimeric forms of hGal-7 was assessed by Western blotting in low-SDSconditions. The control peptide PACAP₂₈₋₃₈ was used in order to ensurethe specificity of hGal-7₍₁₂₉₋₁₃₅₎. Results are representative of threeindependent experiments.

FIG. 10A shows the amino acid sequence of human galectin-7 (NCBIReference Sequence: NP_ 002298.1, SEQ ID NO:14), with the domainscorresponding to residues 13-25, 85-108 and 129-135 being underlined.

FIG. 10B shows the nucleotide sequence of the cDNA encoding humangalectin-7 (NCBI Reference Sequence: NM_ 002307.3, SEQ ID NO:13), withthe coding sequence in bold.

FIG. 11 shows an amino acid sequence alignment of the human prototypicgalectins (galectin-1, 2, 7, 10, 13 and 14). The regions correspondingto residues 13-25, 85-108 and 129-135 of human galectin-7 are indicatedby the brackets. Galectin-1=SEQ ID NO:15; Galectin-2=SEQ ID NO:16;Galectin-10=SEQ ID NO:17; Galectin-13=SEQ ID NO:18; Galectin-14=SEQ IDNO:19).

FIGS. 12A to 12C show the dose-dependent disruption of the hGal-7/Bcl-2heterodimer in the presence of the hGal-7₍₁₂₉₋₁₃₅₎ peptide. FIGS. 12Aand 12B: detection of hGal-7/Bcl-2 heterodimers in the presence of theindicated amounts of recombinant Bcl-2 using an anti-gal-7 antibody(FIG. 12A) or an anti-Bcl-2 antibody (FIG. 12B). FIG. 12C: therecombinant hGal-7 and Bcl-2 (each at 0.5 μM) were incubated withincreasing concentrations of hGal-7₍₁₂₉₋₁₃₅₎ in 20 mM potassiumphosphate buffer (pH 7.1). The effect of hGal-7₍₁₂₉₋₁₃₅₎ on thehomodimerization and heterodimerization (with Bcl-2) of hGal-7 wasassessed by Western blotting in low-SDS conditions with an anti-Gal-7antibody.

DISCLOSURE OF INVENTION

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

The terms “comprising”, “having”, “including”, and “containing” are tobe construed as open-ended terms (i.e., meaning “including, but notlimited to”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All subsets of values within the ranges arealso incorporated into the specification as if they were individuallyrecited herein.

Similarly, herein a general chemical structure with various substituentsand various radicals enumerated for these substituents is intended toserve as a shorthand method of referring individually to each and everymolecule obtained by the combination of any of the radicals for any ofthe substituents. Each individual molecule is incorporated into thespecification as if it were individually recited herein. Further, allsubsets of molecules within the general chemical structures are alsoincorporated into the specification as if they were individually recitedherein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (“e.g.”, “suchas”, etc.) provided herein, is intended merely to better illustrate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed.

Herein, the term “about” has its ordinary meaning. The term “about” isused to indicate that a value includes an inherent variation of errorfor the device or the method being employed to determine the value, orencompass values close to the recited values, for example within 10% or5% of the recited values (or range of values).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

Any and all combinations and subcombinations of the embodiments andfeatures disclosed herein are encompassed by the present invention.

In the studies described herein, the present inventors have shown thatpeptides that target specific domains of galectin-7, more specificallythe dimer interface region corresponding to residues 13-25, 86-102,95-108 and 129-135 of human galectin-7, disrupt the formation ofgalectin-7 dimers and its pro-apoptotic function.

Accordingly, in a first aspect, the present invention provides an agentthat binds to a domain corresponding to residues 13-25, 86-102, 95-108or 129-135 of human galectin-7 and inhibits the dimerization of aprototypic galectin.

The term “prototypic” or “prototypical” galectins refer to galectinsthat form homodimers, consisting of two identical galectin subunits thathave associated with one another. The mammalian galectins that fallunder this category are galectin-1, -2, -5, -7, -10, -11, -13, -14, -15,-16, -17, -19, and -20 (galectin-5, -11, -15, -16, -19, and -20 are notfound in humans).

Human galectin-7 (hGal-7) is a 15 kDa prototype galectin with a singleCRD, monomeric but capable of dimerization in solution. It was firstreported in an effort to identify markers of keratinocytedifferentiation. Galectin-7 involvement in the maintenance of thepluristratified epithelia and epidermal stratification has highlightedits role in wound healing. It was proven to be an efficient growthfactor with therapeutic implications. Some of the more recent advanceson galectin-7 have shown its implication in apoptosis induction invarious types of cell. Galectin-7 expression is induced upon UVradiation and regulated by p53, therefore showing high levels in certaintypes of cancer. hGal-7 has attracted more interest in cancer becauseits preferential expression in epithelial tissues and carcinoma, it isfound in the nucleus of many cancer cells, including hypopharyngeal(HSCCs) and laryngeal (LSCCs) squamous cell carcinomas tissues, coloncarcinoma cells (DLD-1), cervical adenocarcinoma (HeLa), epithelialovarian cancer tissues and oral epithelial dysplasia tissues (Saussez Set al. Histopathology 52: 483-493, 2008; Kuwabara I et al. J Biol Chem277: 3487-3497, 2002; Kim H J et al. Anticancer Res 33: 1555-1561, 2013;de Vasconcelos Carvalho M et al. J Oral Pathol Med 42: 174-179, 2013).Galectin-7 is also observed in the cytosol of colon carcinoma cell line(DLD-1), cervical adenocarcinoma cells (HeLa), epithelial ovarian cancerand oral epithelial dysplasia tissues. (hlen M et al. Nat Biotechnol 28:1248-1250, 2010; Kuwabara I et al. J Biol Chem 277: 3487-3497, 2002; KimH J et al. Anticancer Res 33: 1555-1561, 2013; de Vasconcelos CarvalhoM, et al. J Oral Pathol Med 42: 174-179, 2013.). It is also detected inmitochondrial fractions, most notably in the case of human colorectalcarcinoma and cervical adenocarcinoma cell lines (HCT116, HeLa) and theHaCaT keratinocyte cell line (ViIleneuve C et al. Mol Biol Cell 22:999-1013, 2011). Galectin-7 has been shown to be involved in cancerdevelopment, for example in the growth stimulation of lymphomas (MoisanS, et al., Leukemia. 2003; 17:751-759; Demers M, et al., Cancer Res.2005; 65:5205-5210) and the invasive behavior of ovarian cancer cells(Labrie, M., et al., Oncotarget, 2014. 5(17): p. 7705-21) Galectin-7 wasalso described as a key element in aggressive metastasis following itsoverexpression in breast carcinomas (Demers M, et al., Am J Pathol.2010; 176:3023-3031), and thus represents a potential therapeutictarget. In an embodiment, the agents disclosed herein may be used forthe treatment of any of the diseases/cancers defined above.

The expression “domain corresponding to residues 13-25, 86-102, 95-108or 129-135 of human galectin-7” refers to the domain present any of theprototypic galectins and that corresponds, e.g., based on sequencealignment, to residues 13-25, 86-102, 95-108 or 129-135 of galectin-7.In an embodiment, corresponding domains may be identified for example byaligning the sequences of the different prototypic galectins (see FIG.11). The domains corresponding to residues 13-25, 86-108 (i.e. 86-102and 95-108) or 129-135 of human galectin-7 are indicated by thebrackets. For example, the domain corresponding to residues 129-135 ofhuman galectin-7 in galectin-1 comprises the sequence:Ile-Lys-Cys-Val-Ala-Phe-Asp(128-134).

The expression “inhibits the dimerization of a prototypic galectin”refers to the inhibition of the homodimerization of the prototypicgalectin (e.g., galectin-7), and/or to the heterodimerization of theprototypic galectin (e.g., galectin-7) with other proteins, such asmembers of the bcl-2 family (reference [15]). In an embodiment, thehomodimerization of the prototypic galectin is inhibited. In anotherembodiment, the heterodimerization of the prototypic galectin isinhibited. In another embodiment, both the homodimerization andheterodimerization of the prototypic galectin are inhibited.

The agent includes any compound that binds to a domain corresponding toresidues 13-25, 86-102, 95-108, and/or 129-135 of human galectin-7 andinhibits prototypic galectin (e.g., galectin-7) dimerization. Withoutbeing so limited, such inhibitors include proteins (e.g., dominantnegative, inactive variants), peptides, small molecules, antibodies,antibody fragments, etc.

In an embodiment, the agent that inhibits prototypic galectin (e.g.,galectin-7) dimerization is a neutralizing antibody directed against (orspecifically binding to) to a domain corresponding to residues 13-25,86-102, 95-108 and/or 129-135 of human galectin-7. The term “antibody”or “immunoglobulin” is used in the broadest sense, and covers monoclonalantibodies (including full-length monoclonal antibodies), polyclonalantibodies, humanized antibodies, CDR-grafted antibodies, chimericantibodies, multispecific antibodies, and antibody fragments so long asthey exhibit the desired biological activity (e.g., blocking prototypicgalectin (e.g., galectin-7) dimerization, neutralizing an activityrelated to prototypic galectin (e.g., galectin-7) dimerization).Antibody fragments comprise a portion of a full-length antibody,generally an antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments,diabodies, linear antibodies, single-chain antibody molecules, singledomain antibodies (e.g., from camelids), shark NAR single domainantibodies, and multispecific antibodies formed from antibody fragments.Antibody fragments can also refer to binding moieties comprising CDRs orantigen binding domains including, but not limited to, V_(H) regions(V_(H), V_(H)-V_(H)), anticalins, PepBodies, antibody-T-cell epitopefusions (Troybodies) or Peptibodies. In an embodiment, the antibody is amonoclonal antibody. In another embodiment, the antibody is a humanizedor CDR-grafted antibody.

In general, techniques for preparing antibodies (including monoclonalantibodies and hybridomas) and for detecting antigens using antibodiesare well known in the art (Campbell, 1984, In “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology”, Elsevier Science Publisher, Amsterdam, The Netherlands) and inHarlow et al., 1988 (in: Antibody A Laboratory Manual, CSHLaboratories).

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (s.c.), intravenous (i.v.) or intraperitoneal (i.p.)injections of the relevant antigen (e.g., a polypeptide comprising asequence corresponding to residues 13-25, 86-102, 95-108, and/or 129-135of human galectin-7, or an immunogenic fragment thereof, such as afragment of at least 5, 6, 7, 8, 9 or 10 residues) with or without anadjuvant. It may be useful to conjugate the relevant antigen to aprotein that is immunogenic in the species to be immunized (sometimesreferred to as a carrier), e.g., keyhole limpet hemocyanin, serumalbumin, bovine thyroglobulin, or soybean trypsin inhibitor using abifunctional or derivatizing agent, for example, maleimidobenzoylsulfosuccinimide ester (conjugation through cysteine residues),N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinicanhydride, SOCl₂, or R¹N═C═NR, where R and R¹ are different alkylgroups.

Animals may be immunized against the antigen (a peptide/polypeptidecomprising a sequence corresponding to residues 13-25, 86-102, 95-108,and/or 129-135 of human galectin-7, or an immunogenic fragment thereof,such as a fragment of at least 5, 6, 7, 8, 9 or 10 residues),immunogenic conjugates, or derivatives by combining the antigen orconjugate (e.g., 100 μg for rabbits or 5 μg for mice) with 3 volumes ofFreund's complete adjuvant and injecting the solution intradermally atmultiple sites. One month later the animals are boosted with the antigenor conjugate (e.g., with 1/5 to 1/10 of the original amount used toimmunize) in Freund's complete adjuvant by subcutaneous injection atmultiple sites. Seven to 14 days later the animals are bled and theserum is assayed for antibody titer. Animals are boosted until the titerplateaus. Preferably, for conjugate immunizations, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal antibodies may be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (e.g., U.S. Pat. No. 6,204,023). Monoclonalantibodies may also be made using the techniques described in U.S. Pat.Nos. 6,025,155 and 6,077,677 as well as U.S. Patent ApplicationPublication Nos. 2002/0160970 and 2003/0083293.

In the hybridoma method, a mouse or other appropriate host animal, suchas a rat, hamster or monkey, is immunized (e.g., as hereinabovedescribed) to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the antigen used forimmunization. Alternatively, lymphocytes may be immunized in vitro.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. Thehybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. For example,if the parental myeloma cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

A human chimeric antibody can be produced in the following manner. cDNAencoding heavy chain variable region (V_(H)) and light chain variableregion (V_(L)) obtained from a hybridoma derived from non-human animalcells producing monoclonal antibodies, the cDNA is inserted to each ofexpression vectors for animal cells having DNA encoding a heavy chainconstant region (C_(H)) and light chain constant region (C_(L)) of ahuman antibody so as to construct a human chimeric antibody expressionvector, and this vector is introduced to animal cells to express thehuman chimeric antibody.

A humanized antibody refers to an antibody that is obtained by graftingthe amino acid sequence of the complementary determining region (CDR) ofV_(H) and V_(L) of a non-human animal antibody to CDR corresponding toV_(H) and V_(L) of a human antibody. The region other than CDR of V_(H)and V_(L) is caIled a framework region (hereinbelow, described as “FR”).A humanized antibody can be produced in the following manner. cDNAencoding an amino acid sequence of V_(H) which consists of an amino acidsequence of CDR of V_(H) of a non-human antibody and an amino acidsequence of FR of V_(H) of any human antibody, and cDNA encoding anamino acid sequence of V_(L) which consists of an amino acid sequence ofCDR of V_(L) of a non-human animal antibody and an amino acid sequenceof FR of V_(L) of any human antibody are constructed, these cDNAs areinserted respectively into expression vectors for animal cells havingDNA encoding C_(H) and C_(L) of a human antibody so as to construct ahumanized antibody expression vector, and this vector is inserted intoanimal cells to express the humanized antibody.

Based on the sequences of the human prototypic galectin polypeptides(see FIG. 11), and more particularly of amino acids corresponding toresidues 13-25, 86-102, 95-108, and 129-135 of human galectin-7, theskiIled person would be able to generate antibodies directed againstthis polypeptide/domain(s), which in turn may be used to block itsdimerization and neutralize its activity.

In an embodiment, the agent that inhibits prototypic galectin (e.g.,galectin-7) dimerization is an inactive prototypic galectin peptide orpolypeptide (e.g., dominant negative), or a nucleic acid (e.g., mRNA)encoding such an inactive prototypic galectin peptide or polypeptide,which may compete with the native prototypic galectin (e.g., galectin-7)for dimerization (by binding to the domain(s) corresponding to aminoacids 13-25, 86-102, 95-108 and/or 129-135 of galectin-7), but fails toinduce the signaling cascade and biological activity of the nativeprototypic galectin (e.g., galectin-7) homodimers. Administration of theinactive prototypic galectin (e.g., galectin-7) peptide or polypeptidemay be direct (administration of the polypeptide itself) or indirect,for example via administration of a nucleic acid encoding the inactiveprototypic galectin (e.g., galectin-7) peptide or polypeptide.

In another embodiment, the agent that inhibits prototypic galectin(e.g., galectin-7) dimerization is a peptide or peptidomimetic (or asalt thereof) of 50 residues of less that inhibits human prototypicgalectin (e.g., galectin-7) dimerization, the peptide comprising: (i) adomain comprising at least 5 residues of the sequence of formula IXaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa¹¹-Xaa¹²-Xaa¹³  (I)wherein “-” is a bond; Xaa¹ is L-Ile, D-Ile or an analog thereof; Xaa²is L-Arg, D-Arg or an analog thereof; Xaa³ is L-Pro, D-Pro or an analogthereof; Xaa⁴ is Gly or an analog thereof; Xaa⁵ is L-Thr, D-Thr or ananalog thereof; Xaa⁶ is L-Val, D-Val or an analog thereof; Xaa⁷ isL-Leu, D-Leu or an analog thereof; Xaa⁸ is L-Arg, D-Arg or an analogthereof; Xaa⁹ is L-Ile, D-Ile or an analog thereof; Xaa¹⁰ is L-Arg,D-Arg or an analog thereof; Xaa¹¹ is Gly or an analog thereof; Xaa¹² isL-Leu, D-Leu or an analog thereof; Xaa¹³ is L-Val, D-Val or an analogthereof; or a domain of formula I in which 1 or 2 residue(s) is/aremutated;(ii) a domain comprising at least 5 residues of the sequence of formulaII:Xaa¹⁴-Xaa¹⁵-Xaa¹⁶-Xaa¹⁷-Xaa¹⁸-Xaa¹⁹-Xaa²⁰  (II)wherein “-” is a bond; Xaa¹⁴ is L-Leu, D-Leu or an analog thereof; Xaa¹⁵is L-Asp, D-Asp or an analog thereof; Xaa¹⁶ is L-Ser, D-Ser or an analogthereof; Xaa¹⁷ is L-Val, D-Val or an analog thereof; Xaa¹⁸ L-Arg, D-Argor an analog thereof; Xaa¹⁹ is L-Ile, D-Ile or an analog thereof; andXaa²⁰ is L-Phe, D-Phe or an analog thereof; or a domain of formula II inwhich one of Xaa¹⁴, Xaa¹⁶, Xaa¹⁷, Xaa¹⁸, Xaa¹⁹ or Xaa²⁰ is mutated;(iii) a domain comprising at least 5 residues of the sequence of formulaIII:Xaa²¹-Xaa²²-Xaa²³-Xaa²⁴-Xaa²⁵-Xaa²⁶-Xaa²⁷-Xaa²⁸-Xaa²⁹-Xaa³⁰-Xaa³¹-Xaa³²-Xaa³³-Xaa³⁴-Xaa³⁵-Xaa³⁶-Xaa³⁷-Xaa³⁸-Xaa³⁹-Xaa⁴⁰-Xaa⁴¹-Xaa⁴²-Xaa⁴³  (III)wherein “-” is a bond; Xaa²¹ is L-Phe, D-Phe or an analog thereof; Xaa²²is L-Glu, D-Glu or an analog thereof; Xaa²³ is L-Val, D-Val or an analogthereof; Xaa²⁴ is L-Leu, D-Leu or an analog thereof; Xaa²⁵ is L-Ile,D-Ile or an analog thereof; Xaa²⁶ is L-Ile, D-Ile or an analog thereof;Xaa²⁷ is L-Ala, D-Ala or an analog thereof; Xaa²⁸ is L-Ser, D-Ser or ananalog thereof; Xaa²⁹ is L-Asp, D-Asp or an analog thereof; Xaa³⁰ isL-Asp, D-Asp or an analog thereof; Xaa³¹ is Gly or an analog thereof;Xaa³² is L-Phe, D-Phe or an analog thereof; Xaa³³ is L-Lys, D-Lys or ananalog thereof; Xaa³⁴ is L-Ala, D-Ala or an analog thereof; Xaa³⁵ isL-Val, D-Val or an analog thereof; Xaa³⁶ is L-Val, D-Val or an analogthereof; Xaa³⁷ is Gly or an analog thereof; Xaa³⁸ is L-Asp, D-Asp or ananalog thereof; Xaa³⁹ is L-Ala, D-Ala or an analog thereof; Xaa⁴⁰ isL-Gln, D-Gln or an analog thereof; Xaa⁴¹ is L-Tyr, D-Tyr or an analogthereof; Xaa⁴² is L-His, D-His or an analog thereof, and Xaa⁴³ is L-His,D-His or an analog thereof, or a domain of formula III in which in which1 or 2 residue(s) is/are mutated.

In an embodiment, the peptide comprises a domain comprising at least 5residues of the sequence of formula IIIA or IIIB:Xaa²¹-Xaa²²-Xaa²³-Xaa²⁴-Xaa²⁵-Xaa²⁶-Xaa²⁷-Xaa²⁸-Xaa²⁹-Xaa³⁰-Xaa³¹-Xaa³²-Xaa³³-Xaa³⁴-Xaa³⁵-Xaa³⁶-Xaa³⁷  (IIIA):Xaa³⁰-Xaa³¹-Xaa³²-Xaa³³-Xaa³⁴-Xaa³⁵-Xaa³⁶-Xaa³⁷-Xaa³⁸-Xaa³⁹-Xaa⁴⁰-Xaa⁴¹-Xaa⁴²-Xaa⁴³  (IIIB):

wherein “-” and Xaa²¹ to Xaa⁴³ are as defined above.

As used herein, the term “peptidomimetic” refers to a compoundcomprising a plurality of amino acid residues (naturally- and/ornon-naturally-occurring amino acids, amino acid analogs) joined by aplurality of peptide and/or non-peptide bonds. Peptidomimetics typicallyretain the polarity, three-dimensional size and functionality(bioactivity) of their peptide equivalents, but one or more of thepeptide bonds/linkages have been replaced, often by more stablelinkages. Generally, the bond which replaces the amide bond (amide bondsurrogate) conserves many or all of the properties of the amide bond,e.g. conformation, steric bulk, electrostatic character, potential forhydrogen bonding, etc. Typical peptide bond replacements include esters,polyamines and derivatives thereof as well as substituted alkanes andalkenes, such as aminomethyl and ketomethylene. For example, theabove-mentioned domain or peptide may have one or more peptide linkagesreplaced by linkages such as —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis ortrans), —CH₂SO—, —CH(OH)CH₂—, or —COCH₂—. Such peptidomimetics may havegreater chemical stability, enhanced biological/pharmacologicalproperties (e.g., half-life, absorption, potency, efficiency, etc.)and/or reduced antigenicity relative its peptide equivalent.

The term “amino acid” as used herein includes both L- and D-isomers ofthe naturally occurring amino acids as well as other amino acids (e.g.,naturally-occurring amino acids, non-naturally-occurring amino acids,amino acids which are not encoded by nucleic acid sequences, etc.) usedin peptide chemistry to prepare synthetic analogs of peptides. Examplesof naturally-occurring amino acids are glycine, alanine, valine,leucine, isoleucine, serine, threonine, etc. Other amino acids includefor example non-genetically encoded forms of amino acids, as well as aconservative substitution of an L-amino acid. Naturally-occurringnon-genetically encoded amino acids include, for example, beta-alanine,3-amino-propionic acid, 2,3-diamino propionic acid,alpha-aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine(sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline,t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine,cyclohexylalanine, norleucine (Nle), norvaline, 2-napthylalanine,pyridylalanine, 3-benzothienyl alanine, 4-chlorophenylalanine,2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine,penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid,beta-2-thienylalanine, methionine sulfoxide, L-homoarginine (Hoarg),N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid,2,4-diaminobutyric acid (D- or L-), p-aminophenylalanine,N-methylvaline, homocysteine, homoserine (HoSer), cysteic acid,epsilon-amino hexanoic acid, delta-amino valeric acid, or2,3-diaminobutyric acid (D- or L-), etc. These amino acids are wellknown in the art of biochemistry/peptide chemistry.

The term “analog” when used in reference to an amino acid refers tosynthetic amino acids providing similar side chain functionality (i.e.,structurally similar) as the “native” amino acid and which can besubstituted for an amino acid in the formation of a peptidomimetic.Amino acid analogs include, without limitation, β-amino acids and aminoacids, in which the amino or carboxy group is substituted by a similarlyreactive group or other groups (e.g., substitution of the primary aminewith a secondary or tertiary amine, or substitution of the carboxy groupwith an ester).

For example, aromatic amino acids may be replaced with D- orL-naphthylalanine, D- or L-homophenylalanine, D- or L-phenylglycine, D-or L-2-thienylalanine, D- or L-1-, 2-, 3-, or 4-pyrenylalanine, D- orL-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- orL-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- orL-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine,D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- orL-p-biphenylalanine D- or L-p-methoxybiphenylalanine, D- orL-2-indole(alkyl)alanines, and D- or L-alkylalanines wherein the alkylgroup is substituted or unsubstituted methyl, ethyl, propyl, hexyl,butyl, pentyl, isopropyl, iso-butyl, or iso-pentyl.

Non-carboxylate amino acids can be made to possess a negative charge, asprovided by phosphono- or sulfated (e.g., —SO₃H) amino acids, which areto be considered as non-limiting examples.

Other substitutions may include unnatural alkylated amino acids, made bycombining an alkyl group with a natural amino acid. Basic natural aminoacids such as lysine and arginine may be substituted with alkyl groupsat the amine (NH₂) functionality. Yet other substitutions includenitrile derivatives (e.g., containing a CN-moiety in place of the CONH₂functionality) of asparagine or glutamine, and sulfoxide derivative ofmethionine. In addition, any amide linkage in the peptide may bereplaced by a ketomethylene, hydroxyethyl, ethyl/reduced amide,thioamide or reversed amide moieties, (e.g., (—C═O)—CH₂—),(—CHOH)—CH₂—), (CH₂—CH₂—), (—C═S)—NH—), or (—NH—(—C═O) for (—C═O)—NH—)).

Covalent modifications of the above-mentioned peptide or peptidomimetic(or a salt thereof) are thus included within the scope of the presentinvention. Such modifications may be introduced into the above-mentionedpeptide, peptidomimetic or salt thereof for example by reacting targetedamino acid residues of the peptide with an organic derivatizing agentthat is capable of reacting with selected side chains or terminalresidues. The following examples of chemical derivatives are provided byway of illustration and not by way of limitation.

Cysteinyl residues may be reacted with alpha-haloacetates (andcorresponding amines), such as 2-chloroacetic acid or chloroacetamide,to give carboxymethyl or carboxyamidomethyl derivatives. Histidylresidues may be derivatized by reaction with compounds such asdiethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain, and para-bromophenacyl bromide mayalso be used; e.g., where the reaction is preferably performed in 0.1Msodium cacodylate at pH 6.0. Lysinyl and amino terminal residues may bereacted with compounds such as succinic or other carboxylic acidanhydrides. Other suitable reagents for derivatizingalpha-amino-containing residues include compounds such as imidoesters,e.g., methyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4pentanedione; and transaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin according to known method steps.Derivatization of arginine residues is typically performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group. The specific modification oftyrosinyl residues per se is well-known, such as for introducingspectral labels into tyrosinyl residues by reaction with aromaticdiazonium compounds or tetranitromethane. N-acetylimidazol andtetranitromethane may be used to form O-acetyl tyrosinyl species and3-nitro derivatives, respectively. Tryptophan residues may be methylatedat position 2 (sometimes referred to as 2Me-Trp or Mrp).

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermoreaspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions. Glutaminyl andasparaginyl residues may be frequently deamidated to the correspondingglutamyl and aspartyl residues. Other modifications of theabove-mentioned peptide analog/azasulfurylpeptide may includehydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the alpha-amino groups oflysine, arginine, and histidine side chains acetylation of theN-terminal amine, methylation of main chain amide residues (orsubstitution with N-methyl amino acids) and, in some instances,amidation of the C-terminal carboxyl groups, according to known methodsteps.

Analogs of histidine include those described in Ikeda et al., ProteinEng. (2003) 16 (9): 699-706 (e.g., β-(1,2,3-triazol-4-yl)-DL-alanine),those described in Stefanucci et al., Int. J. Mol. Sci. 2011, 12(5),2853-2890 (aza-histidine, homo-histidine, β²-homo-histidine,β³-homo-histidine, Nor-histidine), N-imidazolyl alanine, methylhistidine, dimethyl histidine, C-triazolyl alanine, histidine methylester, histidinol, and histidinamide.

Analogs of tryptophan includes 2-Me-Trp (or Mrp),5-Methyl-DL-tryptophan, azatryptophan (7-azatryptophan),hydroxytryptophan (5-hydroxytryptophan), fluorotryptophan,aminotryptophan, tryptamine and desaminotryptophan, α-methyl-tryptophan;β-(3-benzothienyI)-D-alanine; β-(3-benzothienyI)-L-alanine;1-methyl-tryptophan; 4-methyl-tryptophan; 5-benzyloxy-tryptophan;5-bromo-tryptophan; 5-chloro-tryptophan; 5-fluoro-tryptophan;5-hydroxy-tryptophan; 5-hydroxy-L-tryptophan; 5-methoxy-tryptophan;5-methoxy-L-tryptophan; 5-methyl-tryptophan; 6-bromo-tryptophan;6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan;6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan;7-methyl tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid;L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid;5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

Analogs of alanine include β-alanine, aminoisobutyric acid (α or β),methylalanine and t-butylalanine.

Analogs of phenylalanine include β-methyl-phenylalanine,β-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine,α-methyl-D-phenylalanine, α-methyl-L-phenylalanine,2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine,2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine,2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine,2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine,2-cyano-L-phenylalanine, 2-fluoro-D-phenylalanine,2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine,2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine,2-nitro-L-phenylalanine, 2,4,5-trihydroxy-phenylalanine,3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine,3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine,3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine,3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine,3-(trifluoromethyl)-D-phenylalanine,3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine,3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine,3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine,3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine,3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine,3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine,3-methyl-D-phenylalanine, 3-methyl-L-phenylalanine,3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine,4-(trifluoromethyl)-D-phenylalanine,4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine,4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine,4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine,4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine,4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine,4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine,4-fluoro-D-phenylalanine, 4-fluoro-L-phenylalanine,4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine,3,3-diphenylalanine.

Analogs of lysine include the related amino acid arginine and analogsthereof such as citrulline; L-2-amino-3-guanidinopropionic acid;L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)₂-OH; Lys(N₃)—OH;Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine;Nω-nitro-L-arginine; α-methyl-ornithine; 2,6-diaminoheptanedioic acid;L-ornithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-D-ornithine;(Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)ethyl)-L-ornithine;(Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-ornithine;D-ornithine; L-ornithine; Arg(Me)(Pbf)-OH; Arg(Me)₂-OH(asymmetrical);Arg(Me)₂-OH (symmetrical); Lys(ivDde)-OH; Lys(Me)₂-OH.HCl; Lys(Me₃)-OHchloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

In embodiments, the domain, peptide or peptidomimetic of the presentinvention include domains, peptides or peptidomimetics with alteredsequences containing substitutions of functionally equivalent amino acidresidues, relative to the above-mentioned domains, peptides orpeptidomimetics. For example, one or more amino acid residues within thesequence can be substituted by another amino acid of a similar polarity(having similar physico-chemical properties) which acts as a functionalequivalent, resulting in a silent alteration. Substitution for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, positively charged (basic)amino acids include arginine, lysine and histidine (as well ashomoarginine and ornithine). Nonpolar (hydrophobic) amino acids includeleucine, isoleucine, alanine, phenylalanine, valine, proline, tryptophanand methionine. Uncharged polar amino acids include serine, threonine,cysteine, tyrosine, asparagine and glutamine. Negatively charged(acidic) amino acids include glutamic acid and aspartic acid. The aminoacid glycine may be included in either the nonpolar amino acid family orthe uncharged (neutral) polar amino acid family. Substitutions madewithin a family of amino acids are generally understood to beconservative substitutions.

The above-mentioned domain, peptide or peptidomimetic (or salt thereof)may comprise only L-amino acids, only D-amino acids, or a mixture of L-and D-amino acids. In an embodiment, the above-mentioned domain, peptideor peptidomimetic (or salt thereof) comprises at least one D-amino acid(e.g., 1, 2, 3, 4, 5 or more D-amino acids). The presence of one or moreD-amino acids typically results in peptides having increased stability(e.g., in vivo) due to decreased susceptibility to protease/peptidasecleavage, but which retain biological activity. In another embodiment,the domain, peptide or peptidomimetic (or salt thereof) comprise onlyL-amino acids.

In embodiments, the above-mentioned peptide or peptidomimetic is in theform of a salt, e.g., a pharmaceutically acceptable salt. As used hereinthe term “pharmaceutically acceptable salt” refers to salts of compoundsthat retain the biological activity of the parent compound, and whichare not biologically or otherwise undesirable. Such salts can beprepared in situ during the final isolation and purification of theanalog, or may be prepared separately by reacting a free base functionwith a suitable acid. Many of the peptides or peptidomimetics disclosedherein are capable of forming acid and/or base salts by virtue of thepresence of amino and/or carboxyl groups or groups similar thereto.

Pharmaceutically acceptable acid addition salts may be prepared frominorganic and organic acids. Representative acid addition salts include,but are not limited to acetate, adipate, alginate, citrate, aspartate,benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, decanoate, digluconate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethansulfonate (isothionate), lactate, maleate,methane sulfonate, nicotinate, 2-naphthalene sulfonate, octanoate,oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate,pivalate, propionate, succinate, tartrate, thiocyanate, phosphate,glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Saltsderived from inorganic acids include hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, and the like. Saltsderived from organic acids include acetic acid, propionic acid, glycolicacid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinicacid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoicacid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonicacid, p-toluene-sulfonic acid, salicylic acid, and the like. Examples ofacids which can be employed to form pharmaceutically acceptable acidaddition salts include, for example, an inorganic acid, e.g.,hydrochloric acid, hydrobromic acid, sulphuric acid, and phosphoricacid, and an organic acid, e.g., oxalic acid, maleic acid, succinicacid, and citric acid.

Basic addition salts also can be prepared by reacting a carboxylicacid-containing moiety with a suitable base such as the hydroxide,carbonate, or bicarbonate of a pharmaceutically acceptable metal cationor with ammonia or an organic primary, secondary, or tertiary amine.Pharmaceutically acceptable salts include, but are not limited to,cations based on alkali metals or alkaline earth metals such as lithium,sodium, potassium, calcium, magnesium, and aluminum salts, and the like,and nontoxic quaternary ammonia and amine cations including ammonium,tetramethylammonium, tetraethylammonium, methylammonium,dimethylammonium, trimethylammonium, triethylammonium, diethylammonium,and ethylammonium, amongst others. Other representative organic aminesuseful for the formation of base addition salts include, for example,ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine,and the like. Salts derived from organic bases include, but are notlimited to, salts of primary, secondary and tertiary amines.

In an embodiment, the above-mentioned peptide or peptidomimetic (or saltthereof) comprises one domain of formula I, II, or III as defined above.In an embodiment, the above-mentioned peptide or peptidomimetic (or saltthereof) comprises two or more (e.g., 2, 3, 4 or 5) domains (repeats) offormula I, II, or III as defined above.

In embodiments, the above-mentioned peptide or peptidomimetic (or saltthereof) may comprise, further to the domain of formula I or II definedabove, one more amino acids (naturally occurring or synthetic)covalently linked to the amino- and/or carboxy-termini of said domain.In an embodiment, the above-mentioned peptide or peptidomimetic (or saltthereof) comprises up to 43 additional amino acids at the N- and/orC-termini to the domain of formula (I), (II), or (I) defined above. Infurther embodiments, the above-mentioned peptide or peptidomimetic (orsalt thereof) comprises up to 40, 35, 30, 25, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 additional amino acidsat the N- and/or C-termini of the domain of formula (I), (II), or (III)defined above. In an embodiment, the above-mentioned peptide orpeptidomimetic (or salt thereof) contains about 45 residues or less, infurther embodiments about 40, 35, 30, 25, 20, 19, 18, 17, 16 or 15residues or less. In an embodiment, the above-mentioned peptide orpeptidomimetic (or salt thereof) contains between about 7 residues toabout 15 residues, for example about 7, 8, 9, 10, 11, 12, 13, 14 or 15residues. In an embodiment, the peptide or peptidomimetic (or saltthereof) comprises, or consists of, 5, 6, 7, 8, 9 or 10 to 20, 25, 30,35, 40, 45, or 50 residues.

In embodiments, the N- and/or C-terminal amino acids of theabove-mentioned peptide or peptidomimetic (or salt thereof) may bemodified, for example by amidation, acetylation, acylation or any othermodifications known in the art.

Accordingly, in another aspect, the present invention provides a peptideor peptidomimetic (or salt thereof) of formula (IV):Z¹-[domain of formula(I),(II), or(III)]-Z²

wherein Z¹ is H (i.e. the peptide or peptidomimetic has a native NH₂terminal) an amino-terminal modification; and Z² is OH (i.e. the peptideor peptidomimetic has a native COOH terminal) or a carboxy-terminalmodification.

In an embodiment, the amino terminal residue (i.e., the free amino groupat the N-terminal end) of the peptide or peptidomimetic (or saltthereof) is modified (e.g., for protection against degradation), forexample by covalent attachment of a moiety/chemical group (Z¹). In anembodiment, the amino-terminal modification (Z¹) is a C₁-C₁₆ or C₃-C₁₆acyl group (linear or branched, saturated or unsaturated), in a furtherembodiment, a saturated C₁-C₆ acyl group (linear or branched) or anunsaturated C₃-C₆ acyl group (linear or branched), in a furtherembodiment an acetyl group (CH₃—CO—, Ac). In another embodiment, thepeptide or peptidomimetic (or salt thereof) has a native NH₂ terminal,i.e. Z¹ is H.

In an embodiment, the carboxy terminal residue (i.e., the free carboxygroup at the C-terminal end of the peptide) of the peptide orpeptidomimetic (or salt thereof) is modified (e.g., for protectionagainst degradation). In an embodiment, the modification is an amidation(replacement of the OH group by a NH₂ group), thus in such a case Z² isa NH₂ group. In a further embodiment, Z² is a sequence of one or moreamino acids (e.g., 1 to 25 additional amino acids, for example 20, 19,18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 aminoacids).

In an embodiment, the peptide (or salt thereof) comprises or consists ofthe following sequence:Ile-Arg-Pro-Gly-Thr-Val-LeU-Arg-Ile-Arg-Gly-Leu-Val-NH₂ (SEQ ID NO:3).Inanother embodiment, the peptide (or salt thereof) comprises or consistsof the following sequence: Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂ (SEQ IDNO:1). In another embodiment, the peptide (or salt thereof) comprises orconsists of the following sequence:Phe-Glu-Val-Leu-Ile-Ile-Ala-Ser-Asp-Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-NH₂(SEQ ID NO:7). In another embodiment, the peptide (or salt thereof)comprises or consists of the following sequence:Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-Asp-Ala-Gln-Tyr-His-His-NH₂ (SEQ IDNO:5).

The peptide or peptidomimetic (or salt thereof) of the present inventionmay further comprise modifications that confer additional biologicalproperties to the peptide or peptidomimetic such as protease resistance,plasma protein binding, increased plasma half-life, intracellularpenetration, etc. Such modifications include, for example, covalentattachment of fatty acids (e.g., C₆-C₁₈) to the peptide orpeptidomimetic, attachment to proteins such as albumin (see, e.g., U.S.Pat. No. 7,268,113); glycosylation, biotinylation or PEGylation (see,e.g., U.S. Pat. Nos. 7,256,258 and 6,528,485). PEGylation may be carriedout using an acylation reaction or an alkylation reaction with areactive polyethylene glycol molecule. Methods for peptide PEGylationare disclosed, for example, in Roberts et al., Chemistry for peptide andprotein PEGylation, Advanced Drug Delivery Reviews, Volume 64,Supplement, December 2012, Pages 116-127). In an embodiment, thepeptide, peptidomimetic or salt thereof is conjugated to a polyethyleneglycol (PEG) chain/moiety (i.e. is PEGylated). The term “PEG chain”refers to polymers of ethylene glycol (represented by the generalformula H(OCH₂CH₂)_(n)OH, where n is an integer of 2, 3, 4, 5, 6, 7, 8,9, or more) which are commercially produced with different molecularweights (e.g., about 200-50,000 Da, 500-40,000 Da, 1000-30,000 Da or2000-10,000 Da). PEGylated peptides/peptidomimetics may be prepared bymodifying certain amino acids in the peptide/peptidomimetic with asuitable group-reactive reagent. For example, a Cys side chain may bemodified with a thiol-reactive agent, or a Lys side chain may bemodified with an amine-reactive agent.

The above description of modification of the peptide or peptidomimetic(or salt thereof) does not limit the scope of the approaches nor thepossible modifications that can be engineered.

The peptide of the invention may be produced by expression in a hostcell comprising a nucleic acid encoding the peptide (recombinantexpression) or by chemical synthesis (e.g., solid-phase peptidesynthesis). Peptides can be readily synthesized by manual and automatedsolid phase procedures well known in the art. Suitable syntheses can beperformed for example by utilizing “t-Boc” or “Fmoc” procedures.Techniques and procedures for solid phase synthesis are described in forexample Solid Phase Peptide Synthesis: A Practical Approach, by E.Atherton and R. C. Sheppard, published by IRL, Oxford University Press,1989. Alternatively, the peptides may be prepared by way of segmentcondensation, as described, for example, in Liu et al., TetrahedronLett. 37: 933-936, 1996; Baca et al., J. Am. Chem. Soc. 117: 1881-1887,1995; Tam et al., Int. J. Peptide Protein Res. 45: 209-216, 1995;Schnolzer and Kent, Science 256: 221-225, 1992; Liu and Tam, J. Am.Chem. Soc. 116: 4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA91: 6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res.31: 322-334, 1988). Other methods useful for synthesizing the peptidesare described in Nakagawa et al., J. Am. Chem. Soc. 107: 7087-7092,1985.

Peptides and peptide analogs comprising naturally occurring amino acidsencoded by the genetic code may also be prepared using recombinant DNAtechnology using standard methods. Peptides produced by recombinanttechnology may be modified (e.g., N-terminal acylation [e.g.,acetylation], C-terminal amidation), using methods well known in theart. Therefore, in embodiments, in cases where a peptide describedherein contains naturally occurring amino acids encoded by the geneticcode, the peptide may be produced using recombinant methods, and may inembodiments be subjected to for example the just-noted modifications(e.g., acylation, amidation). Accordingly, in another aspect, theinvention further provides a nucleic acid encoding the above-mentioneddomain or peptide. The invention also provides a vector comprising theabove-mentioned nucleic acid. In yet another aspect, the presentinvention provides a cell (e.g., a host cell) comprising theabove-mentioned nucleic acid and/or vector. The invention furtherprovides a recombinant expression system, vectors and host cells, suchas those described above, for the expression/production of a peptide ofthe invention, using for example culture media, production, isolationand purification methods well known in the art.

The peptide or peptidomimetic (or salt thereof) of the invention can bepurified by many techniques of peptide purification well known in theart, such as reverse phase chromatography, high performance liquidchromatography (HPLC), ion exchange chromatography, size exclusionchromatography, affinity chromatography, gel electrophoresis, and thelike. The actual conditions used to purify a particular peptide orpeptide analog will depend, in part, on synthesis strategy and onfactors such as net charge, hydrophobicity, hydrophilicity, and thelike, and will be apparent to those of ordinary skill in the art. Foraffinity chromatography purification, any antibody that specificallybinds the peptide or peptidomimetic may for example be used.

In another aspect, the present invention provides a composition (e.g., apharmaceutical composition) comprising the above-mentioned agent (e.g.,peptide, peptidomimetic or salt thereof). In an embodiment, thecomposition further comprises one or more pharmaceutically acceptablecarriers, excipient, and/or diluents.

As used herein, “pharmaceutically acceptable” (or “biologicallyacceptable”) refers to materials characterized by the absence of (orlimited) toxic or adverse biological effects in vivo. It refers to thosecompounds, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with thebiological fluids and/or tissues and/or organs of a subject (e. g.,human, animal) without excessive toxicity, irritation, aIlergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The term “pharmaceutically acceptable carriers, excipient, and/ordiluents” refers to additives commonly used in the preparation ofpharmaceutical compositions and includes, for example, solvents,dispersion media, saline solutions, surfactants, solubilizing agents,lubricants, emulsifiers, coatings, antibacterial and antifungal agents,chelating agents, pH-modifiers, soothing agents, buffers, reducingagents, antioxidants, isotonic agents, absorption delaying agents or thelike (see, e.g., Rowe et al., Handbook of Pharmaceutical Excipients,Pharmaceutical Press; 6^(th) edition, 2009).

The agent that inhibits prototypic galectin (e.g., galectin-7)dimerization (e.g., peptide, peptidomimetic or salt thereof) of thepresent invention may be formulated for administration via anyconventional route, such as intravenous, oral, transdermal,intraperitoneal, subcutaneous, mucosal, intramuscular, intranasal,intrapulmonary, parenteral or topical administration. The preparation ofsuch formulations is well known in the art (see, e.g., Remington: TheScience and Practice of Pharmacy, Lippincott Williams & Wilkins; 21^(st)edition, 2005).

The agent that inhibits prototypic galectin (e.g., galectin-7)dimerization (e.g., peptide, peptidomimetic or salt thereof) may be usedto inhibit any biological, physiological and/or pathological processthat involves a prototypic galectin (e.g., galectin-7) homodimerizationor any biological process that involves a prototypic galectin (e.g.,galectin-7) heterodimerization with other proteins, such as members ofthe Bcl-2 family (reference [15]).

In another aspect, the present invention provides a method (in vitro orin vivo) for inhibiting the dimerization of a prototypic galectin (e.g.,galectin-7), said method comprising contacting said prototypic galectin(e.g., galectin-7) with the agent that inhibits a prototypic galectin(e.g., galectin-7) dimerization (e.g., peptide, peptidomimetic or saltthereof) or the composition described herein. In an embodiment, theabove-mentioned method is for inhibiting the dimerization of aprototypic galectin (e.g., galectin-7) in a cell or in the extracellularspace (since prototypic galectins such as galectin-7 are released bycells via a non-classical secretory pathway). The present invention alsoprovides the use of the agent that inhibits a prototypic galectin (e.g.,galectin-7) dimerization (e.g., peptide, peptidomimetic or salt thereof)or the composition described herein for inhibiting the dimerization of aprototypic galectin (e.g., galectin-7). The present invention alsoprovides the use of the agent that inhibits a prototypic galectin (e.g.,galectin-7) dimerization (e.g., peptide, peptidomimetic or salt thereof)or the composition described herein for the manufacture of a medicamentfor inhibiting the dimerization of a prototypic galectin (e.g.,galectin-7).

Recombinant human galectin-7 has been shown to kill certain types ofcells, such as Jurkat T cells, monocytes and human peripheral T cells([39] and Example 3 below), suggesting that galectin-7 hasimmunosuppressive properties. In another aspect, the present inventionprovides a method for inhibiting galectin-7-mediated apoptosis in acell, said method comprising contacting said cell with the agent thatinhibits galectin-7 dimerization (e.g., peptide, peptidomimetic or saltthereof) or the composition described herein. The present invention alsoprovides the use of the agent that inhibits galectin-7 dimerization(e.g., peptide, peptidomimetic or salt thereof) or the compositiondescribed herein for inhibiting galectin-7-mediated apoptosis in a cell.The present invention also provides the use of the agent that inhibitsgalectin-7 dimerization (e.g., peptide, peptidomimetic or salt thereof)or the composition described herein for the manufacture of a medicamentfor inhibiting galectin-7-mediated apoptosis in a cell. In anembodiment, the above-mentioned cell is an immune cell, such as a Tlymphocyte or a monocyte. In another aspect, the present inventionprovides a method for inhibiting galectin-7-mediated immunosuppressionin a subject, said method comprising administering to said subject aneffective amount of the agent that inhibits galectin-7 dimerization(e.g., peptide, peptidomimetic or salt thereof) or the compositiondescribed herein. The present invention also provides the use of theagent that inhibits galectin-7 dimerization (e.g., peptide,peptidomimetic or salt thereof) or the composition described herein forinhibiting galectin-7-mediated immunosuppression in a subject. Thepresent invention also provides the use of the agent that inhibitsgalectin-7 dimerization (e.g., peptide, peptidomimetic or salt thereof)or the composition described herein for the manufacture of a medicamentfor inhibiting galectin-7-mediated immunosuppression in a subject. In anembodiment, the subject suffers from a galectin-7-expressing cancer.

In another aspect, the present invention provides a method for treatinga prototypic galectin- (e.g., galectin-7) expressing cancer (e.g.,inhibiting tumor growth and/or metastasis) in a subject, said methodcomprising administering to said subject an effective amount of theagent that inhibits prototypic galectin (e.g., galectin-7) dimerization(e.g., peptide, peptidomimetic or salt thereof) or the compositiondescribed herein. The present invention also provides the use of theagent that inhibits prototypic galectin- (e.g., galectin-7) dimerization(e.g., peptide, peptidomimetic or salt thereof) or the compositiondescribed herein for treating a prototypic galectin- (e.g., galectin-7)expressing cancer in a subject. The present invention also provides theuse of the agent that inhibits prototypic galectin (e.g., galectin-7)dimerization (e.g., peptide, peptidomimetic or salt thereof) or thecomposition described herein for the manufacture of a medicament fortreating a prototypic galectin- (e.g., galectin-7) expressing cancer ina subject. In an embodiment, the prototypic galectin- (e.g., galectin-7)expressing cancer is of epithelial origin. In another embodiment, theprototypic galectin- (e.g., galectin-7) expressing cancer is a breastcancer, an ovarian cancer or a lymphoma. In a further embodiment, theprototypic galectin- (e.g., galectin-7) expressing cancer is a breastcancer. In another embodiment, the prototypic galectin- (e.g.,galectin-7) expressing cancer is an ovarian cancer. In anotherembodiment, the prototypic galectin- (e.g., galectin-7) expressingcancer is a lymphoma. In another embodiment, the cancer is a cancer ofneural cells, for example a medulloblastoma.

In another embodiment, the agent that inhibits prototypic galectin(e.g., galectin-7) dimerization could be used to treat other diseaseswhere prototypic galectins (e.g., galectin-7) are involved, for exampleinfectious diseases or diseases/injury of the skin, where galectin-7 isnormally expressed (Gendronneau et al., Mol Biol Cell. 2008 December;19(12):5541-9; Gendronneau et al., PLoS One. 2015 Mar. 5; 10(3):e0119031), in graft rejection (Luo et al., Transplant Proc. 2013 March;45(2):630-4), asthma (Yin et al., Zhonghua Er Ke Za Zhi. 2006 July;44(7):523-6), as well as preeclampsia and miscarriage (Menkhorst et al.,Placenta. 2014 April; 35(4):281-5 and Placenta. 2014 March;35(3):195-201).

The amount of the agent that inhibits agent that inhibits prototypicgalectin (e.g., galectin-7) dimerization (e.g., peptide, peptidomimeticor salt thereof) which is effective for the above-notedactivities/therapeutic uses will depend on several factors including thenature and severity of the disease, the chosen prophylactic/therapeuticregimen, the target site of action, the patient's weight, special dietsbeing followed by the patient, concurrent medications being used, theadministration route and other factors that will be recognized by thoseskiIled in the art. The dosage will be adapted by the clinician inaccordance with conventional factors such as the extent of the diseaseand different parameters from the patient. Typically, 0.001 to 1000mg/kg of body weight/day will be administered to the subject. In anembodiment, a daily dose range of about 0.01 mg/kg to about 500 mg/kg,in a further embodiment of about 0.1 mg/kg to about 200 mg/kg, in afurther embodiment of about 1 mg/kg to about 100 mg/kg, in a furtherembodiment of about 10 mg/kg to about 50 mg/kg, may be used. The doseadministered to a patient, in the context of the present inventionshould be sufficient to effect/induce a beneficial prophylactic and/ortherapeutic response in the patient over time (in the case of a cancer,a decrease in tumor size, inhibition of tumor cell proliferation,increased survival time, etc.). The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration. Effective doses may beextrapolated from dose response curves derived from in vitro or animalmodel test systems. For example, in order to obtain an effective mg/kgdose for humans based on data generated from rat studies, the effectivemg/kg dosage in rat may be divided by six.

In an embodiment, the above-mentioned treatment comprises theuse/administration of more than one (i.e. a combination of)active/therapeutic agent, including the above-mentioned agent thatinhibits prototypic galectin (e.g., galectin-7) dimerization. Thecombination of prophylactic/therapeutic agents and/or compositions ofthe present invention may be administered or co-administered (e.g.,consecutively, simultaneously, at different times) in any conventionaldosage form. Co-administration in the context of the present inventionrefers to the administration of more than one therapeutic in the courseof a coordinated treatment to achieve an improved clinical outcome. Suchco-administration may also be coextensive, that is, occurring duringoverlapping periods of time. For example, a first agent may beadministered to a patient before, concomitantly, before and after, orafter a second active agent is administered. The agents may in anembodiment be combined/formulated in a single composition and thusadministered at the same time. In an embodiment, the one or more activeagent(s) is used/administered in combination with one or more agent(s)or treatment currently used to prevent or treat the disorder in question(e.g., agents or treatments currently used in the treatment of cancers,such as radiotherapy, surgery and/or targeted therapy).

The present inventors have determined that agents that targets specificdomains/residues of galectin-7 that are involved in galectin-7dimerization, more specifically residues 13-25, 86-102, 95-108, and/or129-135 of human galectin-7, may be useful to inhibit biological,physiological and/or pathological processes that involves galectin-7dimerization.

Accordingly, in another aspect, the present invention provides a methodfor determining whether a test agent that may be used to inhibit abiological, physiological and/or pathological process that involvesprototypic galectin (e.g., galectin-7) dimerization, said methodcomprising: contacting a prototypic galectin (e.g., galectin-7)polypeptide with said test agent; and determining whether said testagent binds to a domain corresponding to residues 13-25, 85-102, 95-108,and/or 129-135 of said prototypic galectin (e.g., galectin-7)polypeptide, wherein the binding of said test agent binds to said domainis indicative that said test agent may be used to inhibit a biological,physiological and/or pathological process that involves prototypicgalectin (e.g., galectin-7) dimerization.

In an embodiment, the above-mentioned method further comprisesdetermining whether the test agent inhibits signaling (e.g., activationof a signaling pathway) or activity induced by prototypic galectin(e.g., galectin-7) dimerization in a cell. In an embodiment, theactivity is apoptosis.

Test agents (e.g., drug candidates) that may be screened by themethod/system of the invention may be obtained from any number ofsources including libraries of synthetic or natural compounds. Forexample, numerous means are available for random and directed synthesisof a wide variety of organic compounds and biomolecules, includingexpression of randomized oligonucleotides, peptides, etc. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural or synthetically produced libraries and compounds are readilymodified through conventional chemical, physical and biochemical means.

Screening assay systems may comprise a variety of means to enable andoptimize useful assay conditions. Such means may include but are notlimited to: suitable buffer solutions, for example, for the control ofpH and ionic strength and to provide any necessary components foroptimal activity and stability (e.g., protease inhibitors), temperaturecontrol means for optimal activity and/or stability of galectin-7, anddetection means to enable the detection of the binding of the test agentto galectin-7. A variety of such detection means may be used, includingbut not limited to one or a combination of the following:radiolabelling, antibody-based detection, fluorescence,chemiluminescence, spectroscopic methods (e.g., generation of a productwith altered spectroscopic properties), various reporter enzymes orproteins (e.g., horseradish peroxidase, green fluorescent protein),specific binding reagents (e.g., biotin/(strept)avidin), and others.

The prototypic galectin (e.g., galectin-7) polypeptide used in theabove-noted method may be a full length prototypic galectin (e.g.,galectin-7) polypeptide, or a fragment or variant thereof that comprisesa domain corresponding to residues 13-25, 85-102, 95-108, and/or 129-135of galectin-7.

MODE(S) FOR CARRYING OUT THE INVENTION

The present invention is illustrated in further details by the followingnon-limiting examples.

Example 1: Materials and Methods

Cell Lines and Reagents. The Jurkat cell line was maintained in RPMI1640 medium. The culture medium was supplemented with 10% [v/v] fetalbovine serum, 2 mmol/L of L-glutamine, 10 mM HEPES buffer, and 1 mMsodium pyruvate. All cell culture products/reagents were purchased fromLife Technologies® (Burlington, ON, Canada).

Peptide Synthesis. Peptides were synthesized using standard Fmocchemistry, as previously described [27]. Briefly, all peptides wereassembled using a semi-automatic multi-reactor system. The Rink-amideresin was used as the solid support, and the amino acids of the peptidesequences were introduced under their Fmoc-N-protected form, i.e. 3 eqbased on the original substitution of the resin (0.7 mmol·g⁻¹).Couplings of the protected amino acids were mediated by(Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate(BOP, 3 eq) and N,N-Diisopropylethylamine (DIPEA, 5 eq) in DMF for 1 h.Coupling efficiency was monitored with the qualitative ninhydrin test.Fmoc removal was achieved with 20% piperidine in DMF for 20 min. Inorder to obtain a biotin-conjugated peptide, an ε-amino acid(Fmoc-Ahx-OH) linker was first coupled, as described above, to peptidylresins and then, following the removal of the Fmoc protecting group, aBiotin-NHS derivative (6 eq, AAPPTec) was attached to the peptidylresins with triethylamine (TEA, 6 eq) in dimethylformamide. Peptideswere then deprotected and removed from the resin via an acidolytictreatment with trifluoroacetic acid (TFA) containing 1,2-ethanedithiol(2.5%), phenol (3%) and water (2.5%) for 2 h at room temperature. Thediethyl ether-precipitated crude peptides were purified by preparativeRP-HPLC performed on a Waters® PrepLC 4000 System with a Waters® 2487detector set at 220 nm and an XTerra® Prep MS C₁₈ column. A lineargradient from eluent A to B with 1% B per 2-min increments (EluentA=H₂O, 0.1% TFA, Eluent B=60% CH₃CN/40% H₂O, 0.1% TFA) was used for eachpurification. CoIlected fractions were then analyzed by matrix-assistedlaser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry(Voyager® DE System from Applied Biosystems®) in linear mode using theα-cyano-4-hydroxycinnamic acid matrix (Carlsbad, Calif., USA) andanalytical RP-HPLC with a Phenomenex® Jupiter C₁₈ column to ensure theirhomogeneity. Fractions corresponding to the desired product with puritygreater than 98% were pooled and lyophilized.

Production of recombinant hGal-7 and hGal-1. Human Gal-7 cDNA was clonedinto pET-22b(+) using NdeI and HindIII restriction enzymes. HumanpET-Gal-1 vector was generously donated by Dr. S. Sato (McGillUniversity, QC, Canada). The proteins were produced in E. coli BL21(DE3) cells at 37° C. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (1mM) was added to the bacterial culture at OD_(600 nm)=0.6-0.7 and thenincubated for 4 h at 37° C. to allow protein production. BacterialpeIlets were resuspended in lysis buffer (0.7 mg/mL lysozyme, 10 mM TrispH 8, 100 mM NaCl, 1 mM EDTA, 1 mM DTT and a protease inhibitorcocktail) and then incubated for 1 h at 37° C. prior to centrifugationfor 30 min at 15,000×g (4° C.). The supernatant was then filtered with500 mL bottle top filter (22 μm) (Corning®, New York, N.Y., USA) andthen ran through a lactose-agarose column (Sigma®, St. Louis, Mo., USA).The protein was eluted in 1 mL fractions with 150 mM lactose solution.Purified fractions were analyzed by SDS-PAGE. The hGal-7 was thenconcentrated and purified using Centrifugal filter units (Amicon®Ultra-15, 10K) (EMD®, Millipore®, Etobicoke, ON) in 20 mM potassiumphosphate at pH 7.1. All subsequent experiments with the recombinantproteins were performed in the same buffer solution unless mentionedotherwise. Brilliant Coomassie blue was purchased from BioRad® (Bio-RadLaboratories®, Mississauga, ON, Canada). The recombinant human Bcl-2protein fragment was purchased from Abcam® (Abcam®, Cambridge, UK, Cat #ab73696).

Western Blotting. For the apoptosis tests, whole-cell extracts werehomogenized and resuspended in RIPA buffer (Thermo Fisher Scientific®,Rockford, Ill., USA) containing protease inhibitors (Roche®, Laval, QC,Canada). Equal amounts of whole-cell extracts (25 μg) were separated onSDS-PAGE and transferred onto nitrocellulose membranes (Bio-RadLaboratories®). The membranes were first blocked with 5% milk [w/v] inTBS/0.5% Tween® 20 [v/v] for 1 h at room temperature and subsequentlyblotted overnight in a solution of TBS containing 3% BSA [w/v] and 0.5%Tween 20 [v/v]. The following antibodies were used: a rabbitanti-Poly-(ADP-ribose) polymerase (PARP)-1 (p25) polyclonal antibody(1:5000; Epitomics®, Burlingame, Calif., USA) and a mouse anti-β-actin(1:10000; Sigma-Aldrich®, St-Louis, Mo., USA). Secondary antibodiesconsisted of horseradish peroxidase-conjugated donkey anti-rabbit (GEHealthcare®, Buckinghamshire, England) and sheep anti-mouse (GEHealthcare®) IgG. Detection was performed using the enhancedchemiluminescence method (GE Healthcare®). For the recombinant proteintests, each peptide was dissolved and maintained in 20 mM potassiumphosphate at pH 7.1. The recombinant proteins and peptide dilutions werepre-incubated for 1 h at 4° C. prior to gel migration. The nativepolyacrylamide gel was made without SDS to allow molecular weightdifferentiation between the dimer and monomer. The following antibodieswere used: a goat anti-Gal-7 antibody (1:10000; R&D Systems®,Minneapolis, Minn., USA), a mouse anti-Gal-1 antibody (1:10000;Proteintech®, Chicago, Ill., USA) and a rabbit anti-Bcl-2 antibody(1:1000; Abcam®, Cambridge, UK). Secondary antibodies consisted ofdonkey anti-goat (R&D Systems®) or sheep anti-mouse and sheepanti-rabbit (GE Healthcare®) IgG. Detection was performed as describedabove.

Fluorescent Binding Assay. Recombinant hGal-7 or hGal-1 (10 μg/mL) wascoated overnight at 4° C. on black, flat bottom, 96-well polystyrenemicroplates (Ultident®, Montreal, QC, Canada). Thereafter, the plate wasblocked with Reagent diluent (PBS-BSA 1%) for 1 h, then incubated withunlabeled hGal-7₍₁₂₉₋₁₃₅₎ (cold) for 60 min and finally incubated withbiotin-labeled [Ahx⁰]hGal-7₍₁₂₉₋₁₃₅₎ for 2h. Lastly, StreptavidinR-phycoerythrin (1/500, Jackson Immunoresearch®, West Grove, Pa., USA)was applied to samples for 30 min. All incubations were performed atroom temperature and the washes between incubations were done with 20 mMpotassium phosphate buffer pH 7.1. The plate was read by a Tecan®Infinite M1000 PRO microplate reader at excitation and emissionwavelengths of 488 nm and 670 nm, respectively.

FITC Conjugation and hGal-7 Binding Assay. To assess hGal-7 binding ontoJurkat T cells, 5 μL of a 1.25 mg/mL fluorescein isothiocyanate(FITC)/DMSO solution was added to 300 μL of 1.7 μg/μL recombinant hGal-7in a 0.1 M NaHCO₃ pH 9.2 solution and incubated for 1 h at roomtemperature on a roIler. FITC-labeled hGal-7 was then purified using aPD-10 Sepharose® column (GE healthcare) and eluted with PBS containing0.01% [v/v] sodium azide. FITC-labeled hGal-7 (0.1 μM) was thenpre-incubated with hGal-7₍₁₂₉₋₁₃₅₎ (or related peptides) in 20 mMpotassium phosphate buffer pH 7.1 for 1 h at 4° C. Jurkat cells (5×10⁵cells per sample) were harvested in PBS-0.01% [v/v] sodium azide andincubated for 30 min on ice with the FITC-labeled hGal-7 with andwithout peptides. Cells were then washed with PBS-0.01% [v/v] sodiumazide and resuspended in 400 μL of the same buffer and analyzed on aFACSCalibur® (BD Biosciences®).

Apoptosis Assays with Annexin V/PI Staining. Apoptosis was measured byflow cytometry using FITC-labeled Annexin V (Biolegend®, San Diego,Calif., USA) and propidium iodide. Briefly, the corresponding dilutionsof recombinant hGal-7 and hGal-7₍₁₂₃₋₁₃₅₎ peptides were pre-incubatedfor 1 h at 4° C. in serum-free RPMI 1640 medium. 2.5×10⁵ Jurkat cellswere then harvested in the same medium and incubated with theircorresponding dilutions at 37° C. for 4 h. Cells were washed once in PBSand once in binding buffer (0.01 M HEPES, 0.14 M NaCl, 2.5 mM CaCl₂, pH7.4). Cells were then incubated for 15 min with Annexin V in the dark atroom temperature. A total of 400 μL of binding buffer containing 0.25μg/mL propidium iodide was added to the cells before analysis by flowcytometry.

Statistical analysis. Statistical significance of the experiments wasevaluated using the unpaired Student's t-test or the Fisher's exacttest. Results were considered statistically significant at P≤0.05.

Example 2: Development of Peptides that Inhibit hGal-7 Dimerization

Peptides corresponding to residues 13-25 (hGal-7₍₁₃₋₂₅₎;H-Ile-Arg-Pro-Gly-Thr-Val-Leu-Arg-Ile-Arg-Gly-Leu-Val-Nh₂ (SEQ IDNO:3)), 85-102 (hGal-7₍₅₅₋₁₀₂₎;H-Phe-Glu-Val-Leu-Ile-Ile-Ala-Ser-Asp-Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-NH₂(SEQ ID NO:7)), 95-108 (hGal-7₍₉₅₋₁₀₈₎;H-Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-Asp-Ala-Gln-Tyr-His-His-NH₂ (SEQ IDNO:5)), and 129-135 (hGal-7₍₁₂₉₋₁₃₅₎; H-Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂(SEQ ID NO:1)) of hGal-7, were synthesized and tested for their abilityto inhibit the dimerization of hGal-7, a prototypic galectin. Todetermine whether these peptides could inhibit the formation of thehGal-7 homodimer, recombinant hGal-7 (0.5 μM) was incubated withincreasing concentrations of hGal-7.sub₍₁₃₋₂₅₎, hGal-7₍₈₅₋₁₀₂₎,hGal-7₍₉₅₋₁₀₈₎ or hGal-7₍₁₂₉₋₁₃₅₎ and the formation of homodimers wasmeasured. To measure the ability of the peptides to disrupt theformation of hGal-7 dimers, a mild denaturing (low SDS) native gelelectrophoresis, a commonly used approach to visualize monomer-dimerequilibrium, was used (FIG. 1A and FIG, 6) [32-36]. The results showed aconsistent, decrease of hGal-7 homodimers starting at a 10 μMconcentration of hGal-7₍₁₂₉₋₁₃₅₎, with a saturation dose of 100 μM (FIG.2A). Similar results were obtained with hGal-7₍₁₃₋₂₅₎, hGal-7₍₈₅₋₁₀₂₎,or hGal-7₍₉₅₋₁₀₈₎ but these compounds appeared less potent thanhGal-7₍₁₂₉₋₁₃₅₎ to disrupt hGal-7 homodimers (FIG. 2C). No such effectwas observed using the control peptide PACAP₍₂₈₋₃₈₎, which was selectedbased on similarity in amino acid length and minimal toxicity on thecell line, (FIG. 2A), or on a recombinant human Gal-1 (hGal-1) (FIG.2C), hGal-1 was chosen as a galectin selectivity control since it is aprototype galectin and shares the greatest sequence similarity togalectin-7 (38%) [30]. Moreover, the ability of the hGal-7₍₁₂₉₋₁₃₅₎peptide to disrupt the formation of hGal-7 homodimers was not inhibitedby the presence of lactose (FIG. 2D). Further, the ability ofhGal-7₍₁₂₉₋₁₃₅₎ to bind hGal-7 in a concentration-dependent and specificmanner was further confirmed using a solid phase binding assay. In thisassay, a biotinylated version of hGal-7₍₁₂₉₋₁₃₅₎, still capable ofspecifically inhibiting the formation of hGal-7 homodimers, (FIG. 7) wasused to measure binding on immobilized recombinant hGal-7 (FIG. 3).Again, binding was shown to be specific since biotinylatedhGal-7₍₁₂₉₋₁₃₅₎ could bind hGal-7 and not hGal-1. This specificity atdisrupting hGal-7 dimer formation is provided by distinctthree-dimensional arrangements between otherwise very similar galectinhomologues. Indeed, while all monomeric galectins reveal identicaltopologies, dimer formation in hGal-7 proceeds through a “back-to-back”topology of the monomers and hGal-1 adopts a “side-by-side” structuralarrangement (FIG. 1B) [30]. This structural organization providesadditional means to specifically target and disrupt galectin function.

Example 3: hGal-7₍₁₂₉₋₁₃₅₎ Modulates the Binding of hGal-7 on Jurkat TCells

Galectins are well known for their ability to bind glycosylated cellsurface receptors, most notably on Jurkat T cells, on which galectinbinding induces apoptosis [37-43]. It was thus tested whetherhGal-7₍₁₂₉₋₁₃₅₎ could modulate the binding of hGal-7 on Jurkat T cells,a cell model that is commonly used to test the pro-apoptotic activity ofgalectins [44-46]. For this purpose, recombinant hGal-7 was labeled withfluorescein isothiocyante (FITC) and its binding on the surface ofJurkat T cells was measured by flow cytometry in absence or presence ofincreasing concentrations of hGal-7₍₁₂₉₋₁₃₅₎. The results showed thathGal-7₍₁₂₉₋₁₃₅₎ increased the fluorescent intensity of Jurkat T cells ina concentration-dependent manner following incubation with equal amounts(0.1 μM) of FITC-labeled hGal-7, as compared to fluorescence measured inabsence of peptide (FIG. 4A). No such effect was observed in presence ofa high concentration of the control peptide (PACAP₍₂₈₋₃₈₎). The effectof hGal-7₍₁₂₉₋₁₃₅₎ was specific, statistically significant (FIG. 4B),and consistent with the increased number of monomers, which bind tosurface glycosylated receptors through their CRDs [39]. ThehGal-7₍₁₂₉₋₁₃₅₎ peptide also inhibited the ability of hGal-7 to induceapoptosis in Jurkat T cells, as measured by PARP-1 cleavage (FIG. 5A).No such effect was observed with the control peptide. This effect onapoptosis was confirmed by flow cytometry using Annexin V/PI staining(FIGS. 5B and 5C). Incubation of hGal-7₍₁₂₉₋₁₃₅₎ (or PACAP₍₂₈₋₃₈₎) alonedid not induce apoptosis in Jurkat T cells (FIG. 8).

Example 4: Alanine Scan of hGal 7₍₁₂₉₋₁₃₅₎

To better understand the interaction between hGal-7₍₁₂₉₋₁₃₅₎ and hGal-7.The data obtained using an alanine scan strategy has shown that thesubstitution of the Asp¹³⁰ residue by an Ala moiety abrogates theability of the peptide to disrupt hGal-7 homodimers (Table I and FIG.9). Despite the fact that Asp¹³⁰ does not appear to participate in theformation or stabilization of the wild-type hGal-7 dimer interface (FIG.1C), its replacement to alanine clearly shows significant alteration ofthe hGal-7₍₁₂₉₋₁₃₅₎ potency (FIG. 9). These results suggest a distinctbinding mode between hGal-7₍₁₂₉₋₁₃₅₎ and monomeric hGal-7.

TABLE I Overview of hGal-7₍₁₂₉₋₁₃₅₎ alanine substitute  peptides ActualPeptide Theoretical MW name Sequence MW (g/mol) (g/mol) hGal-L-D-S-V-R-I-F-  849 850.56 7₍₁₂₉₋₁₃₅₎ NH₂ (SEQ ID NO: 2) [Ala¹³⁰]hGal-L-A-S-V-R-I-F-  805.10 805.01 7₍₁₂₉₋₁₃₅₎ NH₂ (SEQ ID NO: 9)[Ala¹³¹]hGal- L-D-A-V-R-I-F-  833.02 833.11 7₍₁₂₉₋₁₃₅₎ NH₂(SEQ ID NO: 10) [Ala¹³³]hGal- L-D-S-V-A-I-F-  772.92 772.27 7₍₁₂₉₋₁₃₅₎NH₂ (SEQ ID NO: 11) [Ala¹³⁵]hGal- L-D-S-V-R-I-A-  763.90 785.617₍₁₂₉₋₁₃₅₎ NH₂ (SEQ ID NO: 12)

Moreover, the modulation of hGal-7 binding on the surface of Jurkat Tcells and an apoptotic response were observed in the presence of thehGal-7₍₁₂₉₋₁₃₅₎ peptide. The increase in fluorescence was themanifestation of the increased hGal-7 binding on cell surface ratherthan its accumulation inside the cell, since the binding assays wereperformed at 0° C. and in the presence of sodium azide (NaN₃), whichwould limit protein internalization [48, 49]. Additionally, the increaseof hGal-7 cell surface binding, seen in the presence of hGal-7₍₁₂₉₋₁₃₅₎,is specific since the control peptide, PACAP₍₂₈₋₃₈₎, did not displaysuch effects. Interestingly, even though an increase of cell surfacehGal-7 binding was observed, a reduction in the ability of the proteinto induce apoptosis of T cells was observed. This supports the idea thatthe increase in hGal-7 binding on cell surface is due to the increasedaccess of the monomer's CRDs binding glycosylated residues on cellsurface receptors while lacking intracellular signaling, highlightingthat effective crosslinking of cell surface receptors is involved in theinduction of apoptosis [25, 50].

Example 5: hGal-7₍₁₂₉₋₁₃₅₎ Modulates the Interaction Between hGal-7 andBcl-2

hGal-7₍₁₂₉₋₁₃₅₎ was tested for its ability to disrupt the interactionbetween hGal-7 and Bcl-2 [15]. As shown in FIG. 12C, the amount ofhGal-7/Bcl-2 heterodimers was decreased in the presence ofhGal-7₍₁₂₉₋₁₃₅₎, especially at the highest dose of 500 μM. These resultsprovide evidence that the hGal-7₍₁₂₉₋₁₃₅₎ interferes not only with thehomodimerization of hGal-7, but also with its heterodimerization withBcl-2.

Although the present invention has been described hereinabove by way ofspecific embodiments thereof, it can be modified, without departing fromthe spirit and nature of the subject invention as defined in theappended claims. The scope of the claims should not be limited by thepreferred embodiments set forth in the examples, but should be given thebroadest interpretation consistent with the description as a whole.

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What is claimed is:
 1. A peptide which is of one of the followingsequences: Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂ (SEQ ID NO:2),Ile-Arg-Pro-Gly-Thr-Val-Leu-Arg-Gly-Leu-Val-NH₂ (SEQ ID NO:4),Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-Asp-Ala-Gln-Tyr-His-His-NH₂ (SEQ IDNO:6) orPhe-Glu-Val-Leu-Ile-Ile-Ala-Ser-Asp-Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-NH₂(SEQ ID NO:8), or a salt thereof.
 2. The peptide or salt thereof ofclaim 1, which is: Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂(SEQ ID NO:2).
 3. Thepeptide or salt thereof of claim 1, whichIle-Arg-Pro-Gly-Thr-Val-Arg-Ile-Arg-Gly-Leu-Val-NH₂ (SEQ ID NO:4). 4.The peptide or salt thereof of claim 1, which is:Asp-Gly-Phe-Lys-Ala-Val-Val-Gly-Asp-Ala-Gln-Tyr-His-His-NH₂ (SEQ NO:6).5. The peptide or salt thereof of claim 1, which is:Phe-Glu-Val-Leu-Ile-Ile-Ala-Ser-Asp-Asp-Gly-Phe-Lys-Ala-Vasl-Val-Gly-NH₂(SEQ ID NO:8).
 6. A method for inhibiting the dimerization of nativehuman galectin-7, said method comprising contacting said native human,galectin-7 with an effective amount of the peptide or salt thereofdefined in claim
 1. 7. The method of claim 6, wherein the peptide orsalt thereof is: Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂ (SEQ ID NO:2).
 8. Amethod for inhibiting galectin-7-mediated apoptosis in, a cell, saidmethod comprising contacting said cell with an effective amount of thepeptide or salt thereof defined in claim
 1. 9. The method of claim 8,wherein the peptide or salt thereof is: Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂(SEQ ID NO:2).
 10. A method for treating a galectin-7-expressing-cancerin a subject, said method comprising administering to said subject aneffective amount of the peptide or salt thereof defined in claim
 1. 11.The method of claim 10, wherein the peptide or salt thereof is:Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂ (SEQ ID NO:2).
 12. The method of claim10, wherein said galectin-7-expressing cancer is a breast cancer, anovarian cancer, or a lymphoma.
 13. The method of claim 12, wherein thepeptide or salt thereof is: Leu-Asp-Ser-Val-Arg-Ile-Phe-NH₂ (SEQ IDNO:2).
 14. The method of claim 12, wherein the galectin-7-expressingcancer is breast cancer.
 15. The method of claim 12, wherein thegalectin-7-expressing cancer is ovarian cancer.
 16. The method of claim12, wherein the galectin-7-expressing cancer is lymphoma.
 17. The methodof claim 13, wherein the galectin-7-expressing cancer is breast cancer.18. The method of claim 13, wherein the galectin-7-expressing cancer isovarian cancer.
 19. The method of claim 13, wherein thegalectin-7-expressing cancer is lymphoma.